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==Procedure== *Embryos are fresh and sitting in 1X PBS in 200ul PCR tube ===Day 1=== #Sterilize bench and tweezers with EtOH, RNaseZap #Make fresh 4% PFA in PBS #*10ml 16% PFA + 4ml 10X PBS + 26ml nfH2O #*Add Argon gas and seal with parafilm to keep it fresh longer #Aspirate PBS from tube and add 200ul 4% PFA #*Incubate at 37C for 15min #Wash twice using cold nf-SSPE #Add 0.25% TX-100 in nf-2XSSPE for 5min at RT #Wash with cold nf-H2O twice #Add 200ul 0.01% pepsin in 0.01N HCl (2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O) for 1min at 37C #*Try 1min, 3min, 5min #Wash with 2ml nf-PBS three times #Prepare 2X Reverse Transcription Mix '''on ice''' {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume''' |- | H2O||157 |- | 10X M-MuLV Buffer||20 |- | 25mM dNTP||2 |- | 4mM aa-dUTP||2 |- | 100uM FISSEQ_RT||5 |- | RNase Inhibitor||2 |- | M-MuLV RTase||10 |- | Total||200 |} #Incubate 10min at 4C and then ~16hr at 37C ===Day 2=== #Wash with 1X PBS once #Add 200ul cold BS(PEG)9 (12ul stock BS(PEG)9 + 588ul 1X PBS prepared on ice) to sample and incubate 1hr at RT #Wash with 1X PBS twice #Add 200ul 1M Tris (pH 8.0) for 30min at RT #Wash with 1X PBS twice #Add RNase H mix and incubate 1hr at 37C #*H2O - 168ul #*RNase H Buffer - 20ul #*Riboshredder - 2ul #*RNase H - 10ul #Wash with nuclease-free H2O twice #Add CircLigase mix and incubate 3hr at 60C #*H2O - 128ul #*CircLigase Buffer 10X - 20ul #*MnCl2 50mM - 10ul #*Betaine 5M - 40ul #*CircLigase II 100U/ul - 2ul #Wash with 1X PBS twice #Add 200ul (1ul 200uM FISSEQ_RCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 60C #Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS #Prepare RCA reaction mix '''on ice''' #*H2O - 174ul #*Phi29 Buffer 10X - 20ul #*dNTP 25mM - 2ul #*aa-dUTP 4mM - 2ul #*Phi29 DNA polymerase 100U/ul - 2ul #Add RCA mix and incubate at 30C overnight (~15hrs) ===Day 3=== #Wash with 1X PBS once #Add 200ul cold BS(PEG)9 (12ul stock BS(PEG)9 + 588ul 1X PBS prepared on ice) to sample and incubate 1hr at RT #Wash with 1X PBS twice #Add 200ul 1M Tris (pH 8.0) for 30min at RT #Wash with 1X PBS twice ====Imaging==== #Add 66ul of 0.5uM FISSEQ_Adpt Cy3 in 2X SSC + 30% formamide pre-heated to 75C #Incubate for 10min at RT #Wash with 2X SSC twice #*Lost 2 embryos, only the largest embryo (#18) left #Mount onto glass slide with 120um spacer between coverslip #*Use microscope mounting medium *552 Laser 1% *Cy3 Gain: 550 *Tilescan 4x5 tiles with 10% overlap *To image whole embryo with max resolution (2k x 2k with 0.3um step size) takes ~5hr
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